Remdesivir resistance in transplant recipients with persistent COVID-19

The medical community currently lacks robust data regarding the incidence, prevalence, and clinical significance of mutations associated with resistance to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) therapeutics. This report describes two renal transplant recipients who, after remdesivir exposure, developed a de novo V792I RNA-dependent RNA polymerase (RdRp) mutation that has recently been found to confer resistance to remdesivir in vitro. To the best of our knowledge, this publication is the first to document the emergence of V792I in patients treated with remdesivir. Our work underscores the critical need for augmented efforts to identify concerning mutations and address their clinical implications.


Introduction
While measures to curb the spread of SARS-CoV-2, a pathogen that has claimed the lives of more than a million Americans, continue to ease in many parts of the United States, the risk of adverse outcomes related to infection in immunocompromised hosts remains substantial 1,2 . In a recent meta-analysis that included nearly 12,000 solid organ transplant (SOT) recipients, vaccination against COVID-19 elicited a humoral response that was signi cantly reduced compared to that of immunocompetent hosts 3 . Though vaccination results in a markedly decreased risk of severe infection in the immunocompetent majority, a diminished response to vaccination translates to a greater risk of severe disease and death in SOT recipients 4 .
Given the substantial risk associated with SARS-CoV-2 infection in SOT recipients, proactive therapy is warranted in patients requiring hospitalization. Remdesivir, a prodrug of the nucleoside analog GS-441524 that inhibits the activity of RdRps in multiple RNA viruses, represents the rst direct-acting antiviral approved by the FDA for the treatment of COVID-19 5 . Very recently, a case of de novo remdesivir resistance was described in a patient who experienced a protracted course of COVID-19 while receiving rituximab and bendamustine for lymphoma 6 . The risk of developing resistance to COVID-19 therapeutics during the course of treatment and the precise prevalence of clinically signi cant mutations in the community are not known.

Case 1
A patient in their 60s with a history of hypertension, diabetes, and vascular disease underwent deceased donor kidney transplant (DDKT) addressing end-stage renal disease (ESRD). Prior to transplant, the patient received two doses of the BNT162b2 vaccine. Initially treated with induction immunosuppression including basiliximab and methylprednisolone, an elevated anti-A titer in the blood type B recipient prompted a transition from basiliximab to anti-thymocyte globulin (ATG). Cytopenias and delayed graft function (DGF) requiring hemodialysis increased suspicion for calcineurin inhibitor (CNI) toxicity, tacrolimus was discontinued, and the patient received an augmented corticosteroid regimen. Ongoing DGF and cytopenias prompted a biopsy of the graft on postoperative day seven, which suggested acute cellular rejection and antibody-mediated rejection. The patient was treated with another dose of ATG, a course of plasmapheresis, and was ultimately transitioned to maintenance immunosuppression including prednisone, mycophenolate, and belatacept. The early postoperative course was further complicated by donor-derived hepatitis C virus (HCV) infection, an intra-abdominal urine leak requiring drainage and nephrostomy tube placement, and pyelonephritis.
Six months after transplant, the patient developed the new onset of malaise, fatigue, cough, and fever. On admission, nasopharyngeal RT-PCR was positive for SARS-CoV-2 with a cycle threshold (Ct) of 27.1.
Genomic sequencing identi ed the B.1.529 (Omicron) subvariant BA.1.1. Not requiring oxygen at that time, the patient received a ve-day course of remdesivir, experienced improvement in symptomatology including defervescence, and was discharged.
24 days after the diagnosis of COVID-19, the patient was readmitted with worsening fatigue, cough, dyspnea, abdominal discomfort, and fever. Nasopharyngeal SARS-CoV-2 RT-PCR was again positive with a Ct of 24.4, and genomic sequencing identi ed Omicron BA.1.1. In the setting of a substantial oxygen requirement, the patient was treated with another ve-day course of remdesivir and a ten-day course of dexamethasone. Genomic sequencing 38 days after COVID-19 diagnosis identi ed the de novo RdRp mutation V792I (G15814A) (Fig. 1). Figure  The patient was treated with multiple cycles of anti-neoplastic therapy that included rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone. Three weeks after the initiation of chemotherapy, the patient developed new ulcerative, bullous, and necrotic cutaneous ndings overlying the renal graft (Fig. 2C). Biopsy of these changes yielded EBV-positive lymphocytes consistent with PTLD. During the course of chemotherapy, the patient experienced severe cytopenias, gastrointestinal bleeding, and hemorrhagic shock. The patient progressed to ESRD, and belatacept and mycophenolate were discontinued. Encountering multiple other complications including cytomegalovirus viremia and bacterial infections over the course of an admission spanning three months, the patient's cough, fever, and hypoxemia all resolved, their skin ndings improved, EBV viremia decreased dramatically, and interval reimaging demonstrated decreased size of the patient's renal graft and associated lymphadenopathy. Three months after the initial diagnosis of COVID-19, IgGs to SARS-CoV-2 nucleoprotein and spike were detectable, the Ct increased to 29.3, and the patient remained free of any symptoms suggesting active respiratory infection. Nasopharyngeal RT-PCR was positive for SARS-CoV-2 with a Ct of 23.3 (Omicron BA.1.1). RT-PCR for other respiratory pathogens was negative. Repeat RT-PCR one week later yielded a Ct of 22.6, and genomic sequencing at that time identi ed a de novo synonymous mutation in RdRp at K890 (Fig. 1). Providers monitored the patient's mild symptoms, which gradually improved over the course of weeks. Ct 153 days after COVID-19 diagnosis performed when the patient was asymptomatic was 34.1. During the patient's prolonged course of infection, two additional de novo non synonymous mutations were also identi ed in nsp6 and orf3 (Supplemental Table 1).

Case 2
A patient in their 50s with a history of vascular disease, splenectomy, and diabetes underwent DDKT addressing ESRD. Prior to transplant, the patient received two doses of the mRNA-1273 vaccine. After receiving induction immunosuppression including methylprednisolone and ATG, the patient was maintained on prednisone, mycophenolate, and tacrolimus. DGF complicating the early postoperative period prompted the empiric administration of methylprednisolone. Graft biopsy performed one week after transplant demonstrated evidence of CNI toxicity, and the patient transitioned from tacrolimus to everolimus. Though graft function improved, the patient experienced a series of complications including medication-induced cytopenias, breakthrough cytomegalovirus viremia, and recurrent bacterial infections over the next several months.
Fourteen months after transplant, the patient developed malaise, shortness of breath, and cough. Nasopharyngeal PCR was positive for SARS-CoV-2, and in the setting of pulmonary in ltrates on x-ray and hypoxemia, the patient received a three-day course of remdesivir and a four-day course of baricitinib.
SARS-CoV-2 genomic sequencing performed on day seven of illness identi ed a de novo V792I (G15814A) mutation in RdRp (Fig. 1). The patient's symptoms improved signi cantly, hypoxemia resolved over the course of a few days, and the patient was discharged. 18 days after COVID-19 was diagnosed, the patient was readmitted with worsening cough and symptomatic hypoxemia. Nasopharyngeal RT-PCR detected SARS-CoV-2 with a Ct of 17.4. X-ray demonstrated worsening patchy in ltrates. Initially managed with methylprednisolone and a ve-day course of remdesivir, the patient developed worsening hypoxemia requiring high-ow oxygen, and CT eventually identi ed multiple cavitary lung lesions (Fig. 2E). An elevated galactomannan level on bronchoalveolar lavage suggested the diagnosis of aspergillosis, and the patient's symptoms and hypoxemia gradually resolved over the course of two weeks with the administration of voriconazole. Of note, de novo mutations in the nsp14 exonuclease ( Fig. 1) and spike protein (Supplemental Table 1) were also identi ed during the course of infection.

Discussion
As the COVID-19 pandemic has progressed, clinicians have increasingly recognized the role that immunosuppression plays in complicating the course of infection. Despite undergoing vaccination prior to transplant, the patient described in Case 1 experienced a protracted course of symptomatic infection that lingered over the course of several months. Similarly striking presentations have occurred in other patients treated with rituximab 6,7 . Compared to other immunosuppressing medications, rituximab appears to be associated with higher rates of adverse outcomes during the course of COVID-19. The increased risk of mortality attributed to rituximab extends to patients treated for rheumatologic and oncologic indications 8 . Like Case 1, the previously vaccinated SOT recipient described in Case 2 also experienced life-threatening complications related to COVID-19 including severe pulmonary aspergillosis.
Sequencing and RT-PCR analysis in both cases strongly suggested the presence of persistent, viable SARS-CoV-2 infection contributing to ongoing symptoms.
When ineffective immune clearance contributes to persistent viral replication in immunocompromised hosts, an increased opportunity for mutation arises. The SARS-CoV-2 non-structural protein, nsp12, encodes the most rapidly replicating RdRp of any known virus. While the exonuclease nsp14 and its cofactor nsp10 offset the low delity of nsp12, the frequency at which SARS-CoV-2 generates mutations remains substantial 9,10 . In the setting of remdesivir exposure and profound immune de cits permitting unchecked replication over an extended period of time, virus isolated from both of the patients in this report independently developed a de novo V792I substitution in ORF1ab, the open reading frame encoding nsp12. A recent study showed that V792I readily develops in vitro after serial cell passage conducted in the presence of increasing remdesivir concentrations 5 . Out of the 11 million genomes deposited in GISAID globally, fewer than 300 isolates contain the V792I substitution 11 . Interestingly, V792I was identi ed in roughly 7% of genomes observed in a recent pre-publication series of immunocompromised patients 12 . Whereas remdesivir incorporated into the elongating RNA of wild-type virus inhibits the integration of a complementary uridine-triphosphate nucleoside, V792I effectively permits a lower uridine-triphosphate concentration to overcome the inhibitory effect of remdesivir. This substitution alone has been demonstrated to increase the remdesivir half-maximal effective concentration (EC50) by 2.6-fold 5 . Other mutations observed in vitro may complement the effect of V792I via distinct mechanisms to further increase the remdesivir EC50 5 . Notably, SARS-CoV-2 isolated throughout the course of infection in both Case 1 and 2 also exhibited the P323L substitution, an Omicron-de ning mutation, in ORF1ab that has been associated with a modest increase in the remdesivir EC50 13 . The combined effect of these mutations and others may limit the clinical e cacy of remdesivir.
Our work emphasizes the potential risk of immune escape in immunocompromised hosts, a scenario in which novel spike mutations evade a suboptimal immune response and contribute to recrudescence of symptomatic infection. Equally concerning are recent reports in immunocompromised hosts highlighting the proclivity of SARS-CoV-2 to develop spike mutations conferring resistance to immunotherapeutics after treatment with monoclonal antibodies 14 .
As remdesivir use has become widespread, and we show that mutations associated with remdesivir resistance arise in vivo, our work emphasizes the importance of augmented surveillance efforts to detect clinically signi cant mutations in immunocompromised patients. Potentially foreshadowing an "endgame" scenario for the COVID-19 pandemic, complex cases like the ones described in this report may presage the eventual need for more advanced molecular diagnostics at the onset of illness to guide therapeutic decisions. It is reasonable to assess for new mutations in patients with prolonged illness who experience persistent infection despite initial therapy.
Finally, the prolonged nature of the infections outlined in this work emphasizes the critical need for thoughtful input from specialists when considering isolation precautions in immunocompromised hosts.
Despite a Ct value approaching 30, detectable anti-SARS-CoV-2 IgG, and markedly improved symptoms three months after COVID-19 diagnosis, the patient described in Case 1 eventually experienced relapsed symptomatic infection associated with a decreasing Ct and newly detected mutations in spike and ORF1ab. Multiple authors have posited that several variants of concern may have originated in immunocompromised hosts 15,16 . The failure to appropriately identify, treat, and control the spread of mutated SARS-CoV-2 isolates could have far-reaching consequences.
Methods SARS-CoV-2 sequencing and bioinformatic analysis. Viral genome sequencing was carried out as described previously 17

Declarations
Ethics/Study approval. This study was approved by the NYULH Institutional Review Board, protocol numbers i21-00493 and i21-00561. Both patients verbally consented to publish their data in a Case Report, including use of the clinical images.

Declaration of Interests.
The authors have no con ict of interest to declare. Independent acquisition of the remdesivir resistance mutation nsp-V792I in two immunocompromised patients.
Full genome mutation pro les of SARS-CoV-2 viruses in longitudinal specimens of two immunocompromised patients treated with remdesivir. Basepair (bp) mutations compared to the Wuhan-Hu-1 reference are shown as ticks, color-coded according to the legend on the lower left. Bp and corresponding amino acid (aa) mutations in nsp12 (RNA-dependent RNA polymerase [RdRp]) and nsp14 (containing 3′-to-5′ exoribonuclease proofreading activity) are labeled and shown in bold if nonsynonymous. The longitudinal acquisition of mutations in nsp12 and 14 are highlighted by colored asterisks. Full genome maps are shown on top. The timeline is shown on the y-axis where time points are indicated on the left and days (d) elapsed since the rst COVID-19 sampling per patient on the right of each plot.
A 3D protein structure of the multi-domain polymerase complex is shown in its active dimeric form. Each domain is colored differently and labeled in the protomer that is shown in ribbon representation, whereas the other domains are shown in sphere representation, respectively. Nsp14 (exonuclease activity) and its co-factor nsp10 convey RNA proofreading in trans and are thus highlighted/shown as ribbons in protomer b together with the other domains in protomer a. The non-synonymous mutations in nsp12 and 14 as well as remdesivir are highlighted and labeled. The structure of the polymerase complex dimer is based on pdb 7egq with remdesivir added by structural overlay of a remdesivir-bound nsp12 complex (pdb 7l1f) 21,22 .

Supplementary Files
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